Characterization of zebrafish Ctla-4. A Alignment of the Ctla-4 homologues from different species generated with ClustalX and Jalview. The conserved and partially conserved amino acid residues in each species are colored in hues graded from orange to red, respectively. The conserved Cysteine residues, and conserved functional motifs, such as B7-binding motif, Tyrosine phosphorylation site, and potential Tyrosine phosphorylation site were indicated separately. The signal peptide, IgV-like domain, transmembrane (TM) domain and cytoplasmic domain were marked at the top of the sequence. B Tertiary protein structures of Ctla-4 ectodomains between humans and zebrafish were predicted by AlphaFold2. The two pairs of disulfide bonds (Cys20-Cys91/Cys46-Cys65 in zebrafish and Cys21-Cys92/Cys48-Cys66 in humans) used to connect the two-layer β-sandwich, and the separate Cys residue (Cys119 in zebrafish and Cys120 in humans) associated with the dimerization of the proteins are indicated. The Cys residues are represented in purple ball-and-stick and the identified or potential B7 binding sites are highlighted in blue. C Dimer of Ctla-4 was identified by Western blot under reducing (+β-ME) or non-reducing (-β-ME) conditions. The ctrl represents the empty control plasmid. The monomer is indicated by one arrow, the dimer by two arrows. D The subcellular localization of Ctla-4 protein was assessed in HEK293T cells transfected with pEGFPN1-Ctla-4 for 48 hours using a two-photon laser-scanning microscope (Original magnification, 630×). The nucleus was stained with DAPI (blue), the cell membrane was stained with DiI (red). E tSNE plots show the relative distribution of common T cell markers cd4-1, cd8a and ctla-4. The data are from splenic single cell RNA sequencing (scRNA-seq) dataset we recently established [59]. F Immunofluorescence staining of lymphocytes separated from blood, spleens, and kidneys of zebrafish. Cells were stained with mouse anti-Ctla-4, together with rabbit anti-Cd4-1 or rabbit anti-Cd8α. DAPI stain shows the location of the nuclei. The images were obtained by two-photon laser-scanning microscope (Original magnification, 630×).

Examination on the IBD-like phenotype in ctla-4-/- zebrafish. A Generation of a homozygous ctla-4-deficient (ctla-4-/-) zebrafish line through CRISPR/Cas9-based knockout of ctla-4 gene on chromosome 9. A 14-bp deletion mutation in exon 2 results in a premature stop at codon 82, which is predicted to produce a defective Ctla-4 protein containing 81 amino acids. B Genotyping of the deficiency of ctla-4 gene by Sanger sequencing. C Knockout efficiency of Ctla-4 selectively examined in spleen and gut tissues of ctla-4-/- zebrafish by Western blot analysis. Gapdh serves as a loading control. D Normal gross appearance of adult wild-type (WT) and ctla-4-/- zebrafish. E Body weight statistics of WT and ctla-4-/- zebrafish (n = 30). F The change of intestine length in WT and ctla-4-/- zebrafish. G The change of splenic size in WT and ctla-4-/- zebrafish. H Representative H & E staining analysis of histopathological changes and quantitation of histology score in anterior, mid and posterior intestines from WT and ctla-4-/- zebrafish. Red arrows indicate mucosal inflammatory cell infiltration, and black arrow indicates transmural inflammatory cell infiltration. I PAS analysis indicates goblet cells in anterior intestine from WT and ctla-4-/- zebrafish. J Quantitation analysis of goblet cells of each villus in the foregut of WT and ctla-4-/- zebrafish (n = 8). K Observation of cell junctions between intestinal epithelial cells in posterior intestines from WT and ctla-4-/- zebrafish under TEM (Hitachi Model H-7650). White triangles indicate tight junctions, black triangle indicates adhesion junctions, and red triangles indicate desmosomes. Data are presented as mean ± standard deviation (SD). Statistical significance was assessed through an unpaired Student’s t test (*p < 0.05; **p <0.01; ***p < 0.001; ****p < 0.0001).

RNA-sequencing analysis of the molecular implications associated with the IBD-like phenotype in ctla-4-/- zebrafish. A Heatmap of different expressed genes between the intestines from wild-type (WT) and ctla-4-/- zebrafish. B Volcano plot showing the up-/down-regulated genes in the intestines of ctla-4-/- zebrafish compared with those of WT zebrafish. Red indicates up-regulated genes and blue indicates down-regulated genes. C GO analysis showing top 10 terms in biological processes of DEGs. D GO analysis showing top 10 terms in biological processes of all up-regulated genes. E Heatmap of representative differently expressed inflammation and chemotaxis-related genes. F The mRNA expression levels of important genes associated with inflammation and chemokines confirmed by real-time qPCR. G Protein-protein interaction network was constructed using the DEGs. The nodes represent the proteins (genes); the edges represent the interaction of proteins (genes). H The MPO activity in the intestines (up) and peripheral blood (down). I Heatmap of IBD biomarker genes and IBD-related genes. J The mRNA expression levels of representative IBD biomarker genes and IBD-related genes were analyzed by real-time qPCR. Data are presented as mean ± standard deviation (SD). Statistical significance was assessed through an unpaired Student’s t test (**p < 0.01; ***p < 0.001; ****p < 0.0001).

Single cell RNA-sequencing analysis of the major cell types associated with the IBD-like phenotype in ctla-4-/- zebrafish. A Classification of cell types from zebrafish intestines by tSNE nonlinear clustering. B Dot plot showing the expression levels of lineage marker genes and percentage of cells per cluster that express the gene of interest. C Expression maps of T cell associated markers within the cell populations of the zebrafish intestines. D Heatmap of the tight/adhesion junction-related genes in enterocytes among the samples from wild-type (WT) and ctla-4-/- zebrafish. E Heatmap of inflammation-related genes involved in cytokine-cytokine receptor interaction in neutrophils among the samples from WT and ctla-4-/- zebrafish. F KEGG enrichment analysis showing the top 15 terms of up-regulated genes in neutrophils from the ctla-4-/- sample versus the WT sample. G KEGG enrichment analysis showing the top 15 terms of up-regulated genes in macrophages from the ctla-4-/- sample versus the WT sample.

Single cell RNA-sequencing analysis of the subset immune-cells associated with the IBD-like phenotype in ctla-4-/- zebrafish. A Classification of subset cells from the T/NK/ILC-like group by tSNE nonlinear clustering. B Dot plot showing the expression levels of subset marker genes and percentage of cells per cluster that express the gene of interest. C Marker gene expression in individual cluster identifying this cluster as ILC3-like cells. D Changes in the composition of subset cells between samples from wild-type (WT) and ctla-4-/- zebrafish. A significantly increased Th2 subset (referred to as Th2 cells 2) in the ctla-4-/- sample was highlighted with a black dashed circle. E Histogram showing the different ratios of subset cells between the WT and ctla-4-/- samples. F Histogram presenting the different numbers of subset cells between the WT and ctla-4-/- samples. G Expression maps of the cytokine il13 within different subset cells between the WT and ctla-4-/- samples. H Dot plots illustrating the differential expression of il13 in T/NK/ILC-like cells from WT and ctla-4-/- zebrafish. I KEGG enrichment analysis showing the top 15 terms of up-regulated genes in the subset of Th2 cells 2. J KEGG enrichment analysis showing the top 15 terms of up-regulated genes in NKT cells. K Scatter plot showing the DEGs of ILC3-like cells in WT and ctla-4-/- zebrafish. The il17a/f1 and il17a/f3 was shown in the scatter plot.

Alteration in microbial composition in the intestines of ctla-4-/- zebrafish. A Venn diagram showing the number of ASVs in zebrafish intestinal microbiota. B Alpha-diversity of microbes was calculated through Shannon index and Simpson index. C Beta-diversity analyzed based on PCoA was shown by using Bray Curtis distance. D The relative abundance of intestinal microbiota at the class level. E-G The relative abundance of Alphaproteobacteria (E), Bacilli (F) and verrucomicrobiae (G) in the intestines from the wild-type (WT) and ctla-4-/- zebrafish. *p < 0.05. H The relative abundance of intestinal microbiota at the family level. I Heatmap analysis of the differential abundances of bacterial communities at family level in the WT and ctla-4-/- zebrafish (p < 0.05). J Cladogram representation of LEfSe analysis showing the differentially abundant bacterial taxa between the intestines from WT (red) and ctla-4-/- (green) zebrafish (p < 0.05).

Examination on the inhibitory function of Ctla-4 in T cell activation. A Assessment of the proliferative activity of T cells from wild-type and ctla-4-/- zebrafish by a mixed lymphocyte reaction combined with PHA-stimulation. The CFSE dilution, which served as an indicator of lymphocyte proliferation, was measured through flow cytometry. B Assessment of the proliferative activity of lymphocytes from ctla-4-/- zebrafish by the administration of sCtla-4-Ig. C Assessment of the proliferative activity of lymphocytes from wild-type zebrafish by supplementing anti-Ctla-4 antibody. D Assessment of the proliferative activity of lymphocytes from ctla-4-/- zebrafish by the administration of sCd28-4-Ig. E Assessment of the proliferative activity of lymphocytes from ctla-4-/- zebrafish by the administration of recombinant Cd80/86 protein. F, G Interactions between Cd80/86 and Cd28 (F), and Cd80/86 and Ctla-4 (G) as predicted by AlphaFold2. On the left are structural models depicting Cd80/86 in complex with Cd28 or Ctla-4. The center panels display per-residue model confidence scores (pLDDT) for each structure, using a color gradient from 0 to 100, where higher scores indicate increased confidence. The right panels show the predicated aligned error (PAE) scores for each model. The well-defined interfaces between Cd28 or Ctla-4 and Cd80/86 are highlighted with red dashed squares. H The interaction between Cd80/86 and Cd28 (left), and Cd80/86 and Ctla-4 (right) were verified by Co-IP. I Binding affinities of the Cd80/86 protein for the Cd28 and Ctla-4 proteins were measured by the MST assay. The KD values are provided. Data are presented as mean ± standard deviation (SD), which were calculated from three independent experiments. Statistical significance was assessed through an unpaired Student’s t test (**p < 0.01; ***p < 0.001; ns denotes no statistical significance).

In vivo inhibition of intestinal inflammation by Ctla-4-Ig. A Percent initial weight of zebrafish after injection of the Ctla-4-Ig or the IgG isotype control. In each group, n = 6. Data show means with SEM analyzed by two-way ANOVA with Sidak’s correction for multiple comparisons. B Representative H&E staining analysis of histopathological changes and quantitation of histology score in the posterior intestine from Ctla-4-Ig or IgG isotype control-supplemented ctla-4-/- zebrafish. Scale bar: 50 μm. C The mRNA expression levels of inflammation-related genes in the Ctla-4-Ig or IgG isotype control treatment groups of ctla-4-/- zebrafish. D The mRNA expression levels of IBD biomarker genes and IBD-related genes in the Ctla-4-Ig or IgG isotype control treatment groups of ctla-4-/- zebrafish. The p value was generated by unpaired two-tailed Student’s t-test. **p < 0.01; ***p < 0.001; ****p < 0.0001.

The organization, sequence and phylogenetic analysis of zebrafish ctla-4 gene. A Comparison of the intron/exon organizations of ctla-4 gene in zebrafish and humans. Exons and introns are shown with black boxes and lines, and their size are indicated by the numbers found above and below the sequences respectively. B The nucleotide and amino acid sequences of ctla-4 gene and Ctla-4 protein. The underline indicates the signal peptide, the circles represent the conserved cysteine residues. C Phylogenetic analysis of the relationship of Ctla-4 between zebrafish and other species. The unrooted phylogenetic tree was constructed through the neighbor-joining method based on the amino acid alignment (ClustalX) of amino acid sequences. The numbers represent the percentage bootstrap confidence derived from 500 replicates.

Preparation of mouse anti-Ctla-4 antibody. A SDS-PAGE detection of the recombinant Ctla-4 protein with extracellular domain (ECD). Lane 1, 2 and 3 represent the protein markers, blank, and target protein, respectively. B Western blot analysis of the mouse anti-EGFP and anti-Ctla-4 antibodies that bind to the recombinant Ctla-4-EGFP fusion proteins expressed in HEK293T cells. C Western blot analysis of native Ctla-4 protein in zebrafish intestinal tissues using mouse anti-Ctla-4 antibody.

Examination on the functional genes and pathways associated with the IBD-like phenotype in ctla-4-/- zebrafish. A Top 5 KEGG enrichment bar plot of up-regulated genes in ctla-4-/- zebrafish intestines versus wild-type zebrafish intestines. B Top 10 KEGG enrichment bar plot of down-regulated genes in ctla-4-/- zebrafish intestines versus wild-type zebrafish intestines. C, D Changes in the expression of genes associated with lymphocyte chemotaxis, positive regulation of ERK1/ERK2 cascades, Calcium and MAPK signaling pathways in the ctla-4-/- zebrafish intestines analyzed by using a collection of pre-defined gene sets retrieved from GO (C) and KEGG (D) database. The p value, false discovery rates (FDR) and normalized enrichment score (NES) are shown above each pathway graph.

Examination on the involvement of apoptotic process in epithelial cells and expression of inflammation-related genes in neutrophils and B cells in the intestines of ctla-4-/- zebrafish. A Expression map of the epithelial markers within the cell populations of the zebrafish intestines. B Expression map of the neutrophil markers within the cell populations of the zebrafish intestines. C KEGG enrichment bar plot of all DEGs from epithelial cells. D Heatmap of inflammation-related genes in B cells from wild-type and ctla-4-/- intestines.

Examination on the activation of T cell subsets in the intestines of ctla-4-/- zebrafish. A Marker gene expression in individual cluster identifying the cluster as NKT cells. B Heatmap of up-regulated genes involved in cytokine-cytokine receptor interaction in NKT cells among the wild-type and ctla-4-/- samples. C KEGG enrichment analysis showing top 15 terms of up-regulated genes in Cd8+ T cells in ctla-4-/- samples versus wild-type samples. D Heatmap of up-regulated genes involved in cytokine-cytokine receptor interaction in Cd8+ T cells among the wild-type and ctla-4-/- samples.

Preparation of recombinant proteins and examination on the molecular interactions. A-C SDS-PAGE detection of the purified recombinant soluble Ctla-4-Ig (A) and Cd28-Ig (B) proteins and the Cd80/86 extracellular domain (ECD) (C) with Coomassie brilliant blue staining. D Flow cytometry analysis of the interaction between Cd80/86 and Cd28 (up), as well as Cd80/86 and Ctla-4 (down). Data are presented as mean ± SD, which were calculated from three independent experiments. Statistical significance was evaluated using an unpaired Student’s t test (**p < 0.01; ***p < 0.001).